Artificial chromosome constructs containing foreign nucleic acid sequences

ABSTRACT

The invention provides artificial chromosome constructs containing foreign nucleic acid sequences, such as viral nucleic acid sequences, and methods of using these artificial chromosome constructs for therapy and recombinant virus production.

BACKGROUND OF THE INVENTION

This invention relates to artificial chromosome constructs containingforeign nucleic acid sequences, such as viral nucleic acid sequences,and methods of using these constructs for therapy and for recombinantvirus production.

Artificial chromosomes are large, DNA-based vectors that have been usedextensively in the construction of DNA libraries for complex genomemapping and analysis. Artificial chromosomes have been derived fromyeast (yeast artificial chromosomes: YACs), bacteria (bacterialartificial chromosomes: BACs, and P1-derived artificial chromosomes:PACs), and mammals (mammalian artificial chromosomes: MACs), such ashumans (human artificial chromosomes: HACs). These vectors includeelements derived from chromosomes that are responsible for replicationand maintenance, and are capable of stably maintaining large genomic DNAfragments.

Herpes Simplex virus (HSV) is the prototypic human herpes virus. Despitethe fact that HSV is a human pathogen, there has been a great deal ofinterest in using HSV as a therapeutic agent. The HSV genome has beensequenced, and many HSV mutants have been generated and usedspecifically in this context. Generation of HSV mutants has been carriedout by using drug selection or by co-transfection of cells with plasmidDNA, usually modified by insertion of a marker gene, and intact viralDNA. Mutants are identified by screening for either drug resistance orrecombination and expression of the marker gene, or by plaquehybridization. Another method that has been used to generate herpesvirus mutants involves the use of cosmid sets that, taken together,contain complete herpes virus genomes. For example, cosmid sets thatcontain the entire genomes of Pseudorabies virus (PRV), Varicella-Zostervirus (VZV), Herpes Simplex virus (HSV), Cytomegalovirus (CMV), andEpstein-Barr virus (EBV) have been created. In constructing completeviral genomes from these cosmids, viral sequences are released from thecosmid backbones and transfected into cells. Viral plaques are producedvia recombination between the overlapping fragments, which togetherrepresent the entire genome. Specific mutations are made in the viralgenomes by manipulating the cosmid DNA.

SUMMARY OF THE INVENTION

The invention provides artificial chromosome constructs containingforeign nucleic acid sequences, such as viral nucleic acid sequences,and methods of using these artificial chromosome constructs for therapy(e.g., gene therapy) and recombinant virus production.

Accordingly, in one aspect, the invention features an artificialchromosome construct containing a nucleic acid sequence that directsformation of a recombinant virus (e.g., a lytic or a non-lytic virus)upon introduction into a cell. Optionally, the artificial chromosomeconstruct, either in the artificial chromosome portion or in the nucleicacid sequence portion, further includes a heterologous nucleic acidsequence that, for example, encodes a therapeutic gene product, such asa growth factor, a hormone, an enzyme, a vaccine antigen, a cytotoxin,an immunomodulatory protein, an antisense RNA molecule, or a ribozyme.The artificial chromosome portion of the construct can be derived from abacterial artificial chromosome, a P1-derived artificial chromosome, ayeast artificial chromosome, or a mammalian (e.g., human) artificialchromosome. The recombinant virus encoded by the nucleic acid sequenceincluded in the artificial chromosome construct can be a herpes virus,such as a herpes simplex virus. Other viruses that can be encoded by thenucleic acid sequence are listed below.

In another aspect, the invention features a method of producing arecombinant virus in a cell, for example, a cell in a mammal, in whichan artificial chromosome construct as described above is introduced intothe cell. This method can further involve introducing into the cell anamplicon that is packaged into a recombinant virion upon introduction ofthe artificial chromosome construct into the cell.

The invention also provides a method of introducing a heterologousnucleic acid sequence into a cell, for example, a cell in a mammal, inwhich an artificial chromosome construct as described above isintroduced into the cell.

Also included in the invention is a method of killing a cell, forexample, a cell in a mammal (e.g., a cancer cell), in which anartificial chromosome construct as described above is introduced intothe cell.

The invention also features a cell having an artificial chromosomeconstruct stably integrated into its genome. In one example, theartificial chromosome construct includes a nucleic acid sequence thatencodes an HSV genome in which an immediate early gene is mutated ordeleted (see below). The invention also includes methods of making thesecells.

The invention provides many advantages. For example, when bacterialartificial chromosome constructs are employed in the invention, theconstructs can be easily and efficiently propagated in bacteria. This isparticularly advantageous in the case of constructs including viralgenes that would be cytopathic if the constructs were propagated inmammalian cells. This is also advantageous in the manufacturing ofrecombinant viruses, because large-scale, bacterial culture methods canbe used, rather than methods employing mammalian cell culture. Anadditional advantage of the invention is that, in contrast tocosmid-based systems for virus production (see above), which rely onrecombination of several molecules to reconstitute a complete viralgenome, an entire viral genome can be contained in a single artificialchromosome construct, providing increased efficiency and geneticstability. As is discussed further below, this is particularlyadvantageous in the trans packaging of amplicons into recombinantviruses using the methods of the invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the HindIII fragment of plasmidBAC-TK. Fragments AB and CD correspond to nucleotides 47860-47150 and47057-45582, respectively, of the HSV-1 strain 17 genome (Genbankaccession numbers X14112, O00317, O00374, and S40593). “H” representsHindIII restriction sites, and “cm” and “tk” represent chloramphenicoland thymidine kinase coding sequences, respectively.

FIG. 2 is a graph showing one-step growth curves for HSV-BAC, r25, andr26 (see Experimental Results, below).

FIG. 3A is a schematic representation of a method for carrying outHSV-BAC mutagenesis. Briefly, homologous recombination is carried outbetween HSV-BAC and a construct containing (i) sequences complementaryto UL53 (“53seq”), (ii) a kanamycin gene (“kan”), and (iii) sequencescomplementary to UL55 (“55seq”), resulting in the UL54 gene, which ispresent in the HSV genome between the UL53 and UL55 genes, beingreplaced by the kanamycin gene.

FIG. 3B is a schematic representation of PCR analysis that can be usedto characterize clones in which the homologous recombination eventdescribed above in reference to FIG. 3A has taken place.

FIG. 4 is a photograph of a gel showing the results of an experiment inwhich the stability of the BAC plasmid p25 was compared to the stabilityof cosmid 14 (Cunningham et al., Virology 197(1):116-124, 1993).

DETAILED DESCRIPTION

The invention provides artificial chromosome constructs that containforeign nucleic acid sequences, such as viral nucleic acid sequences.Optionally, the artificial chromosome constructs also containheterologous nucleic acid sequences, i.e., nucleic acid sequences thatare not naturally a component of the artificial chromosome or the viralnucleic acid sequences. The artificial chromosome constructs of theinvention can be used in methods for producing recombinant viruses incells in vivo, for example, in therapy methods (e.g., gene therapymethods), or in vitro, for example, in recombinant virus productionmethods or in amplicon packaging.

Artificial chromosomes into which foreign nucleic acid sequences can beinserted for use in the invention include, for example, bacterialartificial chromosomes (BACs, e.g., pBeloBAC11 or pBAC108L; see, e.g.,Shizuya et al., Proc. Natl. Acad. Sci. USA 89(18):8794-8797, 1992; Wanget al., Biotechniques 23(6):992-994, 1997), P1-based artificialchromosomes (PACs), yeast artificial chromosomes (YACs; see, e.g.,Burke, Gcnct. Anal. Tech. Appl. 7(5):94-99, 1990), and mammalianartificial chromosomes (MACs; see, e.g., Vos, Nat. Biotechnol.15(12):1257-1259, 1997; Ascenzioni et al., Cancer Lett. 118(2):135-142,1997), such as human artificial chromosomes (HACs).

Viral nucleic acid sequences that can be inserted into artificialchromosomes to generate the artificial chromosome constructs of theinvention can be derived from any of a number of well known viruses,such as viruses that include a circular replication intermediate. Forexample, members of DNA virus families, e.g., the Herpeseviridae (e.g.,HSV-1, HSV-2, VZV, CMV, EBV, HHV6, or HHV7), Adenoviridae, Poxviridae,Papovaviridae (e.g., papillomaviruses and polyomaviruses), Parvoviridae,and Hepadnaviridae families, can be used. Members of RNA virus families,the genomes of which can be made into DNA by standard moleculartechniques, can also be used. For example, members of the Coronaviridae,Picornaviridae, Retroviridae, Caliciviridae, Togaviridae (e.g.,flaviviruses), and Astroviridae families, which are single, plusstranded viruses, can be used. Also, members of the Paramyxoviridae,Orthomyxoviridae, Filoviridae, Rhabdoviridae, Arenaviridae, andBunyaviridae families, which are single, negative stranded viruses, canbe used. Double stranded RNA viruses, such as those of the familyReoviridae, can also be used in the invention.

As is discussed above, viral nucleic acid sequences are included in theartificial chromosome constructs of the invention, so that recombinantviruses are produced from the constructs upon introduction into cells.In some applications of the invention, it is desirable that therecombinant virus produced in this manner results in killing of thecell. In this case, the virus produced from the artificial chromosomeconstruct can be a virus that kills the cell in which it is produced by,for example, inducing lysis or apoptosis of the cell. This is desirable,for example, if the cell is a cancer cell, such as a cancer cell of anervous-system type tumor, for example, an astrocytoma,oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,Schwannoma, neurofibrosarcoma, or medulloblastoma cell. Other types oftumor cells that can be killed, pursuant to the present invention,include, for example, melanoma, pancreatic cancer, prostate carcinoma,breast cancer, lung cancer, colon cancer, gastric cancer, fibrosarcoma,squamous cell carcinoma, neurectodermal, thyroid tumor, pituitary tumor,lymphoma, hepatoma, mesothelioma, and epidermoid carcinoma cells.

Other therapeutic applications in which killing of a target cell isdesirable include, for example, ablation of keratinocytes and epithelialcells responsible for warts, ablation of cells in hyperactive organs(e.g., thyroid), ablation of fat cells in obese patients, ablation ofbenign tumors (e.g., benign tumors of the thyroid or benign prostatichypertrophy), ablation of growth hormone-producing adenohypophysealcells to treat acromegaly, ablation of mammotropes to stop theproduction of prolactin, ablation of ACTH-producing cells to treatCushing's disease, ablation of epinephrine-producing chromaffin cells ofthe adrenal medulla to treat pheochromocytoma, and ablation ofinsulin-producing beta islet cells to treat insulinoma.

This effect can be augmented if the artificial chromosome construct alsocontains a heterologous nucleic acid sequence encoding one or more of,for example, a cytotoxin, an immunomodulatory protein (i.e., a proteinthat either enhances or suppresses a host immune response to anantigen), or a tumor antigen. Examples of immunomodulatory proteinsinclude, e.g., cytokines (e.g., interleukins, for example, any ofinterleukins 1-15, α-, β-, or y-interferons, tumor necrosis factor,granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), and granulocyte colony stimulatingfactor (G-CSF)), chemokines (e.g., neutrophil activating protein (NAP),macrophage chemoattractant and activating factor (MCAF), RANTES, andmacrophagc inflammatory peptides MIP-1a and MIP-1b), complementcomponents and their receptors, immune system accessory molecules (e.g.,B7.1 and B7.2), adhesion molecules (e.g., ICAM-1, 2, and 3), andadhesion receptor molecules. Examples of tumor antigens that can beproduced using the present methods include, e.g., the E6 and E7 antigensof human papillomavirus, EBV-derived proteins (Van der Bruggen et al.,Science 254:1643-1647, 1991), mucins (Livingston et al., Cur. Opin.Immun. 4(5):624-629, 1992), such as MUC1 (Burchell et al., Int. J.Cancer 44:691-696, 1989), melanoma tyrosinase, and MZ2-E (Van derBruggen et al., supra). (Also see WO 94/16716 for a further descriptionof modification of viral vectors to include genes encoding tumorantigens or cytokines.) The heterologous nucleic acid sequence can beinserted into either the artificial chromosome portion of the constructor into the viral portion of the construct in any of these examples.

In other applications of the methods of the invention, it is desirablethat a recombinant virus, produced upon introduction of an artificialchromosome construct of the invention into a cell, does not kill thecell. These applications include, for example, use of artificialchromosome constructs that contain a heterologous gene encoding atherapeutic gene product, such as a growth factor, a hormone, a vaccineantigen, an antisense RNA molecule, or a ribozyme (see below). Theseapplications also include using artificial chromosome constructs toimmunize against a virus encoded by the nucleic acid sequence includedin the construct. It may be desirable in these applications that thevirus produced from the artificial chromosome construct is attenuated ormutated so that it does not replicate and/or so that it cannot kill thecell in which it is produced by, for example, inducing lysis orapoptosis of the cell. Numerous appropriate mutant viruses having thesecharacteristics are known and can readily be adapted for use in theinvention by those of ordinary skill in the art. For example, in thecase of HSV, the vectors of Geller (U.S. Pat. No. 5,501,979; WO90/09441; American Type Culture Collection (ATCC), Rockville, Md., ATCCAccession Number 40544), Breakfield (EP 453,242-A1), Speck (WO96/04395), Preston et al. (WO 96/04394), DeLuca (U.S. Pat. No.5,658,724), and Martuza (U.S. Pat. No. 5,585,096) can be adapted for usein such methods. Specific examples of attenuated HSV mutants that can beused include HF (ATCC VR-260), MacIntyre (ATCC VR-539), MP (ATCCVR-735); HSV-2 strains G (ATCC VR-724) and MS (ATCC VR-540); as well asmutants having mutations in one or more of the following genes: theimmediate early genes ICP0, ICP4, ICP22, ICP27, and ICP47 (U.S. Pat. No.5,658,724); genes necessary for viral replication, VL9, VL5, VL42, DNApol, and ICP8; the γ34.5 gene; the ribonucleotide reductase gene; theVP16 gene (i.e., Vmw65, WO 91/02788; WO 96/04395; WO 96/04394); and thegH, gL, gD or gB genes (WO 92/05263, 94/21807, 94/03207).

An appropriate therapeutic product to be encoded by a heterologousnucleic acid sequence included in this type of artificial chromosomeconstruct (i.e., a construct that results in production of a non-lyticvirus upon introduction into a cell) can readily be selected by oneskilled in the art, depending on the desired result. For example, theheterologous gene product can be a protein, such as a growth factor(e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor(NGF), VGF, or VEGF), an enzyme, a hormone, or a vaccine antigen.Specific examples of protein gene products that can be produced in theinvention include, e.g., tyrosine hydroxylase 1, 2, or 3, which can beused in the treatment of Parkinson's disease; Nerve Growth Factor (NGF,e.g., the NGF γ subunit), which can be used in the treatment ofParkinson's disease; hypoxanthine-guanine phosphoribosyl transferase,which can be used in the treatment of Lesch-Nyhan disease;β-hexosaminidase α-chain, which can be used in the treatment ofTay-Sachs and Sandhoff's diseases; Human Immunodeficiency Virus (HIV)nef, which can be used in the treatment of the neurological symptoms ofHIV, and insulin, which can be used in the treatment of diabetes. Otherprotein gene products that can be produced using the methods of theinvention include, for example, signal transduction enzymes, e.g.,Protein Kinase C; transcription factors, e.g., c-fos, NF-Kβ; oncogenes,e.g., erbB, erbB-2/ncu, and ras; neurotransmitter receptors, e.g.,glutamate receptor, dopamine receptor, and adenosine deaminase receptor(WO 92/10564, WO 89/12109, EP 0 420 911); and the cystic fibrosisprotein (WO 91/02796, WO 92/05273, and WO 94/12649).

The therapeutic product can also be an RNA molecule, such as anantisense RNA molecule that, by hybridization interactions, can be usedto block expression of a cellular or pathogen mRNA. Alternatively, theRNA molecule can be a ribozyme (e.g., a hammerhead or a hairpin-basedribozyme) designed either to repair a defective cellular RNA, or todestroy an undesired cellular or pathogen-encoded RNA (see, e.g.,Sullenger, Chem. Biol. 2(5):249-253, 1995; Czubayko et al., Gene Ther.4(9):943-949, 1997; Rossi, Ciba Found. Symp. 209:195-204, 1997; James etal., Blood 91(2):371-382, 1998; Sullenger, Cytokines Mol. Ther.2(3):201-205, 1996; Hampel, Prog. Nucleic Acid Res. Mol. Bio. 58:1-39,1998; Curcio et al., Pharmacol. Ther. 74(3):317-332, 1997).

The components of the artificial chromosome constructs of the inventioncan be assembled using standard methods. For example, as is describedfurther below, a viral sequence can be inserted into an artificialchromosome by co-transfection of cells with (i) a construct containingthe artificial chromosome, flanked by sequences homologous to regions ofthe viral genome, and (ii) the viral genome, so that recombination takesplace in the cells, resulting in production of a recombinant virusincluding the artificial chromosome. Also as is described further below,recombinant DNA molecules can be isolated from cells in which anartificial chromosome construct and viral nucleic acid sequences havebeen co-transfected, leading to production of a recombinant virus byhomologous recombination, and the isolated DNA molecule can bemanipulated to insert or delete a gene, as desired. Direct cloningmethods, employing unique sites in the viral genome and the artificialchromosome, can also be used to assemble the components of theartificial chromosome constructs of the invention.

The location within an artificial chromosome into which viral orheterologous nucleic acid sequences are inserted will vary depending,for example, on whether disruption of a particular viral function isdesired. For example, if it is desired that the artificial chromosomeconstruct includes viral sequences that will not lead to the productionof replicable virus in cells (see above), then the artificial chromosomeand the viral sequences can be combined so that a nucleic acid sequencethat is essential for viral replication is disrupted. Similarly,insertion of a heterologous nucleic acid sequence can be used to disruptan essential viral gene. Examples of essential genes in HSV, forexample, are described above.

Alternatively, it may be desirable to have the artificial chromosomeconstruct include viral sequences that will lead to the production of areplicable virus in cells (see above). In this case, recombinationbetween the artificial chromosome and the viral nucleic acid sequence togenerate an artificial chromosome construct (or introduction of aheterologous nucleic acid sequence) can be carried out in anon-essential region of the virus genome. For example, in the case ofHSV, such an insertion can be in the thymidine kinase gene (UL23, seebelow; this insertion can be made alone or in combination with anotherinsertion), in any one or more of the following genes: RL1, RL2 (i.e.,IE 110), the locus encoding latency associated transcripts, UL2 (theuracil-DNA glycosylase gene), UL3, UL4, UL10, UL11, UL13, UL16, UL20,UL24, UL40 (the small subunit of ribonucleotide reductase), UL41 (virionhost shut-off factor), UL43, UL44, UL45, UL46, UL47, UL50, UL55, UL56,USI (IE68), US2, US3 (the protein kinase gene), US4 (the glycoprotein Ggene), US5, US7 (the glycoprotein I gene), USS (the glycoprotein Egene), US9, US 10, US11, and US 12 (IE12), or in an intergenic sequence.

A heterologous nucleic acid sequence can be inserted into an artificialchromosome construct in a location that renders it under the control ofa regulatory sequence of the artificial chromosome or viral nucleic acidsequences. Alternatively, the heterologous nucleic acid sequence can beinserted as part of an expression cassette that includes regulatoryelements, such as promoters or enhancers. Appropriate regulatoryelements can be selected by one of ordinary skill in the art based on,for example, the desired tissue-specificity and level of expression. Forexample, a cell-type specific or tumor-specific promoter can be used tolimit expression of a gene product to a specific cell type. This isparticularly useful, for example, when a cytotoxic, immunomodulatory, ortumor antigenic gene product is being produced in a tumor cell in orderto facilitate its destruction. In addition to using tissue-specificpromoters, local administration of an artificial chromosome constructcan result in localized expression.

Examples of non-tissue specific promoters that can be used in theinvention include the early Cytomegaloviris (CMV) promoter (U.S. Pat.No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al.,Molec. Cell Biol. 5:281, 1985). Also, HSV promoters, such as HSV-1 IEand IE 4/5 promoters, can be used.

Examples of tissue-specific promoters that can be used in the inventioninclude, for example, the desmin promoter, which is specific for musclecells (Li et al., Gene 78:243, 1989; Li et al., J. Biol. Chem. 266:6562,1991; Li et al., J. Biol. Chem. 268:10403, 1993); the enolase promoter,which is specific for neurons (Forss-Petter et al., J. Neuroscience Res.16(1):141-156, 1986); the β-globin promoter, which is specific forerythroid cells (Townes et al., EMBO J. 4:1715,1985); the tau-globinpromoter, which is also specific for erythroid cells (Brinster et al.,Nature 283:499, 1980); the growth hormone promoter, which is specificfor pituitary cells (Behringer et al., Genes Dev. 2:453, 1988); theinsulin promoter, which is specific for pancreatic beta cells (Selden etal., Nature 321:545, 1986); the glial fibrillary acidic proteinpromoter, which is specific for astrocytes (Brenner et al., J. Neurosci.14:1030, 1994); the tyrosine hydroxylase promoter, which is specific forcatecholaminergic neurons (Kim et al., J. Biol. Chem. 268:15689, 1993);the amyloid precursor protein promoter, which is specific for neurons(Salbaum et al., EMBO J. 7:2807, 1988); the dopamine β-hydroxylasepromoter, which is specific for noradrenergic and adrenergic neurons(Hoyle et al., J. Neurosci. 14:2455, 1994); the tryptophan hydroxylasepromoter, which is specific for serotonin/pineal gland cells (Boularandet al., J. Biol. Chem. 270:3757, 1995); the choline acetyltransferascpromoter, which is specific for cholinergic neurons (Hersh et al., J.Neurochem. 61:306, 1993); the aromatic L-amino acid decarboxylase (AADC)promoter, which is specific for catccholaminergic/5-HT/D-type cells(Thai et al., Mol. Brain Res. 17:227, 1993); the proenkephalin promoter,which is specific for neuronal/spermatogenic epididymal cells (Borsooket al., Mol. Endocrinol. 6:1502, 1992); the reg (pancreatic stoneprotein) promoter, which is specific for colon and rectal tumors, andpancreas and kidney cells (Watanabe et al., J. Biol. Chem. 265:7432,1990); and the parathyroid hormone-related peptide (PTHrP) promoter,which is specific for liver and cecum tumors, and neurilemoma, kidney,pancreas, and adrenal cells (Campos et al., Mol. Rnfovtinol. 6:1642,1992).

Examples of promoters that function specifically in tumor cells includethe stromelysin 3 promoter, which is specific for breast cancer cells(Basset et al., Nature 348:699, 1990); the surfactant protein Apromoter, which is specific for non-small cell lung cancer cells (Smithet al., Hum. Gene Ther. 5:29-35, 1994); the secretory leukoproteaseinhibitor (SLPI) promoter, which is specific for SLPI-expressingcarcinomas (Garver et al., Gene Ther. 1:46-50, 1994); the tyrosinasepromoter, which is specific for melanoma cells (Vile et al., GeneTherapy 1:307, 1994; WO 94/16557; WO 93/GB1730); the stress induciblegrp78/BiP promoter, which is specific for fibrosarcoma/tumorigenic cells(Gazit et al., Cancer Res. 55(8):1660, 1995); the AP2 adipose enhancer,which is specific for adipocytes (Graves, J. Cell. Biochem. 49:219,1992); the α-1 antitrypsin transthyretin promoter, which is specific forhepatocytes (Grayson et al., Science 239:786, 1988); the interleukin-10promoter, which is specific for glioblastoma multiform cells (Nitta etal., Brain Res. 649:122, 1994); the c-erbB-2 promoter, which is specificfor pancreatic, breast, gastric, ovarian, and non-small cell lung cells(Harris et al., Gene Ther. 1:170, 1994); the α-B-crystallin/heat shockprotein 27 promoter, which is specific for brain tumor cells (Aoyama etal., Int. J. Cancer 55:760, 1993); the basic fibroblast growth factorpromoter, which is specific for glioma and meningioma cells (Shibata etal., Growth Fact. 4:277, 1991); the Epidermal Growth Factor Receptorpromoter, which is specific for squamous cell carcinoma, glioma, andbreast tumor cells (Ishii et al., Proc. Natl. Acad. Sci. USA 90:282,1993); the mucin-like glycoprotein (DF3, MUC1) promoter, which isspecific for breast carcinoma cells (Abe et al., Proc. Natl. Acad. Sci.USA 90:282, 1993); the mts1 promoter, which is specific for metastatictumors (Tulchinsky et al., Proc. Natl. Acad. Sci. USA 89:9146, 1992);the NSE promoter, which is specific for small-cell lung cancer cells(Forss-Petter et al., Neuron 5:187, 1990); the somatostatin receptorpromoter, which is specific for small cell lung cancer cells(Bombardieri et al., Eur. J. Cancer 31A: 184, 1995; Koh et al., Int. J.Cancer 60:843, 1995); the c-erbB-3 and c-erbB-2 promoters, which arespecific for breast cancer cells (Quin et al., Histopathology 25:247,1994); the c-erbB4 promoter, which is specific for breast and gastriccancer cells (Rajkumar et al., Breast Cancer Res. Trends 29:3, 1994);the thyroglobulin promoter, which is specific for thyroid carcinomacells (Mariotti et al., J. Clin. Endocrinol. Meth. 80:468, 1995); theα-fetoprotein promoter, which is specific for hepatoma cells (Zuibel etal., J. Cell. Phys. 162:36, 1995); the villin promoter, which isspecific for gastric cancer cells (Osborn et al., Virchows Arch. A.Pathol. Anat. Histopathol. 413:303, 1988); and the albumin promoter,which is specific for hepatoma cells (Huber, Proc. Natl. Acad. Sci. USA88:8099, 1991).

As noted above, the artificial chromosome constructs of the inventioncan be used in in vivo methods, for example, to introduce a therapeuticgene product into a cell, or to kill a cell, either directly orindirectly, through a lytic viral intermediate. To carry out thesemethods, the artificial chromosome constructs can be administered by anyconventional route used in medicine. As general guidance, an artificialchromosome construct of the invention can be administered directly intothe tissue in which an effect, e.g., expression, is desired, forexample, by direct injection or by surgical methods (e.g., stereotacticinjection into a brain tumor; Pellegrino et al., Methods inPsychobiology (Academic Press, New York, N.Y., 67-90, 1971)). Anadditional method that can be used to administer artificial chromosomeconstructs into the brain is the convection method described by Bobo etal. (Proc. Natl. Acad. Sci. USA 91:2076-2080, 1994) and Morrison et al.(Am. J. Physiol. 266:292-305, 1994). In the case of tumor treatment, asan alternative to direct tumor injection, surgery can be carried out toremove the tumor, and the artificial chromosome constructs of theinvention inoculated into the resected tumor bed to ensure destructionof any remaining tumor cells.

Alternatively, the construct can be administered via a parenteral route,e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal,intraepidermal, or intramuscular route, or via a mucosal surface, e.g.,an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, orurinary tract surface. An artificial chromosome construct formulated inassociation with bupivacaine (see below) is advantageously administeredinto muscle tissue. When a neutral or anionic liposome or a cationiclipid, such as DOTMA or DC-Chol (see below), is used, the formulationcan be advantageously injected via intravenous, intranasal(aerosolization), intramuscular, intradermal, or subcutaneous routes. Anartificial chromosome construct in a naked form can advantageously beadministered via intramuscular, intradermal, or subcutaneous routes.

Any of a number of well known formulations for introducing nucleic acidmolecules into cells in mammals can be used in the invention. (See,e.g., Remington's Pharmaceutical Sciences (18^(th) edition), ed. A.Gennaro, 1990, Mack Publishing Co., Easton, Pa.) For example, theartificial chromosome constructs can be used in a naked form, free ofany packaging or delivery vehicle. The artificial chromosome constructscan be simply diluted in a physiologically acceptable solution, such assterile saline or sterile buffered saline, with or without a carrier.Artificial chromosome constructs can also be administered in calciumphosphate solutions.

The artificial chromosome constructs can be also associated with agentsthat facilitate cellular uptake of nucleic acid molecules. For example,the artificial chromosome constructs can be complemented with a chemicalagent that modifies cellular permeability, such as bupivacaine (see,e.g., WO 94/16737), encapsulated into a liposome, or associated withcationic lipids or silica, gold, or tungsten microparticles.

Anionic and neutral liposomes are well known in the art (see, e.g.,Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for adetailed description of methods for making liposomes) and are useful fordelivering a large range of products, including nucleic acid molecules.Cationic lipids are also well known and are commonly used for genedelivery. Such lipids include Lipofectin™, also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives, such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid, such as DOPE (dioleylphosphatidylethanolamine), as, for example, described in WO 90/11092.

Other transfection-facilitating compounds can be added to formulationscontaining cationic liposomes. A number of these compounds are describedin, e.g., WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. Theyinclude, e.g., spermine derivatives useful for facilitating thetransport of DNA through the nuclear membrane (see, for example, WO93/18759) and membrane-permeabilizing compounds such as GALA,Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for gene delivery, asdescribed in WO 91/359, WO 93/17706, and Tang et al. (Nature 356:152,1992). In this case, the microparticle-coated nucleic acid molecules canbe injected via intradermal or intraepidermal routes using needlelessinjection devices (“gene gun”), such as those described in U.S. Pat. No.4,945,050, U.S. Pat. No. 5,015,580, and WO 94/24263.

The amount of artificial chromosome construct to be administereddepends, e.g., on the specific goal to be achieved, the strength of anypromoter used in the construct, the condition of the mammal intended foradministration (e.g., the weight, age, and general health of themammal), the mode of administration, and the type of formulation. Ingeneral, a therapeutically or prophylactically effective dose of, e.g.,from about 1 ng to about 1 mg, preferably, from about 10 μg to about 800μg, is administered to human adults. The administration can be achievedin a single dose or repeated at intervals.

In addition to being used in therapeutic methods, the artificialchromosome constructs of the invention can be used to cheaply andefficiently produce recombinant viruses in vitro. As is discussed above,prior viral production methods have employed cosmid sets containingportions of viral genomes that are recombined to generate complete viralgenomes upon introduction into cells. This method can result ininstability of the viral genome. The artificial chromosome constructs ofthe invention by-pass the need for recombination between multiplecosmids, leading to enhanced stability of the produced viral genome. Inaddition, the artificial chromosome constructs of the invention, such asthose including BACs, can be propagated in host systems, such asbacteria, that are not adversely affected by the presence of viral genesthat are harmful to mammalian cells. Standard methods can be used forintroducing mutations into such constructs or for introducingheterologous genes to be included in a recombinant viral vector.

The artificial chromosome constructs of the invention also enableefficient packaging of amplicons into recombinant viral particles (see,e.g., WO 97/05263). Amplicons are plasmids containing a gene (e.g., anyof the heterologous nucleic acid sequences described above) under thecontrol of regulatory sequences (e.g., any of the promoters or enhancersdescribed above and, optionally, sequences that direct processing ofmRNA, such as a polyadenylation sequence), a viral origin ofreplication, and viral packaging signals. As is described further belowin reference to HSV, when an amplicon is co-transfected into a cell withan artificial chromosome construct of the invention, which includesviral DNA that results in recombinant virus formation upon introductioninto a cell, the amplicon is packaged in a virion, thus providing aneffective gene delivery vector. Recombinant viruses produced using thesemethods can be used, for example, as viral vectors for therapy, e.g.,gene therapy.

The artificial chromosome constructs of the invention can also be usedin methods for generating cell lines that can be used, for example, forvirus production. As a specific example, an HSV-BAC construct can bemade in which any one or any combination of the immediate early genes(i.e., ICP0, ICP4, ICP22, ICP27, and ICP47) is mutated or deleted, usingstandard methods, such as those described above. Optionally, a geneencoding a selectable marker, such as an antibiotic resistance gene(e.g., a neomycin resistance gene), can be inserted into the viralgenome in a non-essential region (see above) or into the BAC. Thisconstruct can then be transfected into cells (e.g., Vero cells, seeabove) using standard methods, and clones containing integrated HSV canbe selected by culturing the cells in the presence of, for example, theantibiotic. Cells produced in this manner do not, without furthermanipulation, produce virus, due to the missing immediate early gene(s).Thus, the cells can be cultured in the absence of the cytotoxicity thatcan be associated with viral production. When it is desired to inducevirus production in these cells, the missing immediate early gene(s) canbe introduced into the cells by transfection.

These methods provide significant advantages over prior methods which,in requiring the simultaneous introduction of multiple, large plasmidsinto a single cell, are inefficient and expensive. In addition, it isstraightforward to carry out deletion or mutation of genes propagated inbacteria, as is possible when using the bacterial artificial chromosomeconstructs of the invention. In contrast, attempting to accomplish thisusing cell lines is extremely difficult, if not impossible. Finally,although described above in reference to HSV-BAC, these methods can bereadily adapted to employ the use of any of the artificial chromosomeconstructs described above.

Experimental Results

Generation of Recombinant Viruses and BAC Plasmids

We used bacterial artificial chromosome (BAC) technology to clone theentire HSV genome. A plasmid, BAC-TK, was constructed with viral tksequences flanking the signals necessary for chromosomal maintenance inbacteria and the chloramphenicol resistance gene (FIG. 1). This plasmidwas linearized and co-transfected with HSV-1 infectious DNA into Verocells (ATCC CRL 1587). The resultant virus stocks were screened with 100μM acyclovir (ACV), and resistant viruses were isolated and plaquepurified. Southern blot and PCR analysis confirmed insertion of BAC andchloramphenicol sequences within the tk locus. The recombinant virus wasdesignated HSV-BAC-TK.

Vero cells were infected for 2 hours with HSV-BAC-TK at an m.o.i. of 3,harvested, and DNA was extracted as is described below in Materials andMethods. Isolated circular DNA was electroporated into E. coli andrecombinant colonies were screened for resistance to chloramphenicol.Bacteria that were resistant to the antibiotic were subjected to furtheranalysis. Briefly, DNA was extracted from 11 colonies and subjected toPCR analysis using primer sets corresponding to HSV genes US6, UL10,UL30, and UL40. All 11 clones tested were positive for all four primersets. DNA was extracted from 8 of the 11 clones and digested with EcoRI,EcoRV, and HindIII. The results showed that the entire genome of HSV hadbeen cloned.

Rescue of Infectious Virus Pro-Geny from Hsv-Bac Clones

Two clones from the original 8, p25 and p26, were selected for furtheranalysis. Transfection of these BAC plasmids resulted in plaquesformation after two days. Rescued virus was amplified, and the viral DNAwas extracted and digested with restriction endonucleases. Therestriction endonuclease patterns of rescued viruses r25 and r26 wereidentical to those of p25, p26, and HSV-BAC-TK. To determine whether therescued viruses behaved similarly to the parental virus, all threeviruses were tested in a one step growth curve (FIG. 2). The resultsshow that there is little or no difference in the growth rates of theparental input virus and the bacterially rescued viruses over time.

Construction and Characterization of an HSV UL54 Mutant

To illustrate the power of bacterial genetics in the construction of HSVmutants, the essential HSV gene, UL54, can be deleted as follows. (SeeFIGS. 3A and 3B.) The gene product of the UL54 gene, ICP27, plays manyroles in HSV infection. The protein, which is essential for viralgrowth, is found predominantly in the nucleus, where it inhibitspre-mRNA splicing, interacts with the host cell's small nuclearribonucleoprotein particles, binds to RNA, prevents thenucleocytoplasmic transport of intron-containing mRNAs, and regulateslate-gene protein synthesis by facilitating the export of late viralRNAs.

To clone the flanking sequences for homologous recombination, two primersets can be used. One set binds to a sequence that lies immediatelyupstream of the UL54 coding sequence and to a sequence about 1 kbupstream from this site, i.e., in the UL53 gene. The second set binds tosequences immediately 3′ of the UL54 stop codon and to a sequence about1 kb downstream from this site, i.e., in the UL55 gene. PCR fragmentsgenerated using these primers can be cloned into placi, generatingp53-laci-55. Kanamycin coding sequences can be amplified by PCR andcloned in frame with the α-complementation lacZ sequences ofp53-placi-55 to create p53-laci-kan-UL55. Homologous recombinationbetween this plasmid and BAC DNA results in replacement of the HSV UL54coding sequences with the laci promoter driving the α-complementationlacZ/kanamycin fusion sequences. p53-placi-kan-55 sequences can bereleased from the plasmid backbone, gel purified, and transformed intocompetent E. coli that contain p25. Recombinants can then be screenedfor their ability to grow on kanamycin, IPTG, and chloramphenicolplates. Southern hybridization can be used to confirm the presence ofthe mutation.

A clone having the correct mutation, which could be designated pΔUL54,could be further analyzed, as follows. The growth kinetics of rΔUL54,the rescued virus from pΔUL54, r25, HSV-BAC-TK, and wild type HSV strainF could be compared in 2—2 and Vero cells. 2—2 cells are a stable cellline that constitutively express ICP27, and thus are able to complementthe UL54 defect. All four viruses would be expected to replicate to wildtype levels in 2—2 cells, but rΔUL54 would not be expected to grow onVero cells. Restriction endonuclease patterns of DNA extracted from therescued mutant viruses or from input plasmid DNA could be carried out toconfirm that BAC plasmids remain stable during the mutagenesisprocedure.

HSV-BAC DNA Packages Amplicons

Vectors based on HSV have been used extensively for gene transfer.HSV-plasmid vectors (amplicons) contain a eukaryotic expression cassetteand the HSV signals for replication and packaging. The amplicon vectoris replicated and packaged into virions when co-propagated with a helpervirus, either wild type virus, replication-defective virus, or virusgenerated from HSV cosmids. In fact, a modified cosmid set has been usedto produce amplicon preparations that are free of contaminating helpervirus (Fraefel et al., supra). However, herpes virus cosmids are proneto deletion and rearrangement and, further, are often heterogeneous innature. A BAC clone of HSV overcomes these problems.

To assess the stability of HSV-BAC clones, as compared to HSV cosmidclones, we purified and digested DNA isolated from bacterial culturesgrown continuously for either 1, 3, or 7 days. (See FIG. 4 for theresults obtained on samples taken at days 1 and 7.) The restrictionendonuclease pattern of p25 remained unchanged throughout this period,while the restriction endonuclease pattern of cosmid 14 (Cunningham etal., sapra) was altered drastically. Heterogeneity in the bacterialpopulation was appraised by streaking out E. coli containing either p25or cosmid 48 (Cunningham et al., supra) and mini-prepping 5 clones each.The results showed that E. coli containing BAC clones are homogeneous,while E. coli containing cosmid clones are heterogeneous in nature.Thus, maintenance of HSV sequences in BACs is preferable for theproduction of homogenous, infectious viral DNA for therapeutic purposes.

Trans functions were tested by determining the ability of HSV-BAC DNA topackage amplicon plasmid DNA. An amplicon plasmid containing the GreenFluorescent Protein (GFP) gene under the control of the IE 4/5 promoter,or an amplicon plasmid containing a lacZ gene under the control of theRous Sarcoma Virus (RSV) promoter (pHSVLaC), was co-propagated witheither transfected p25, HSV cosmid DNA (6, 28, 14, 56, and 48;Cunningham et al., supra), or infectious F strain viral DNA (ATCCVR-733). Cells were harvested after four days and infectious particleswere titered by infecting fresh monolayers and counting either plaques,green cells, or blue cells (Table 2). The results showed that HSV-BACDNA could package amplicons more efficiently than cosmid DNA andinfectious viral DNA. These data show that HSV-BAC, with appropriatemodifications, can be used as a powerful vector for gene transfer.

Materials and Methods

Viruses and Cells

Vero and Vero 2—2 cells were propagated and assayed for drugsusceptibility as described by Coen et al. (Virology 168:221-231, 1989).Virus titers were determined in triplicate on Vero cells. Recombinantviruses were plaque-purified two times and the mutations were confirmedby Southern blot hybridization and PCR analysis.

Plasmids

Plasmid pTK-AB was created by subcloning the Bg1II/EcoRI fragment frompXhoIf (the XhoI F fragment of HSV-1 strain 17 cloned into plasmidpAT153) into the BamHI/EcoRI sites of pHSV1ac (Geller et al., stpra).The tk-containing plasmid, BH13 (Horsburgh et al., Cell 86(6):949-959,1996) was digested with PstI and XhoI, to release the 3′ end of the tkgene, and the fragment was subcloned into the PstI/XhoI site of pSC1180, creating plasmid TK-CD. Plasmids TK-AB and TK-CD were digestedwith HindIII/MscI and HindIII/Sa1I, respectively, and the resultingfragments were subcloned into the HpaI/Sa1I sites of pBeloBAC (Dr. H.Shizuya and Dr. M. Simon, Department of Biology, California Institute ofTechnology, Pasadena, Calof.) to create plasmid pBAC-TK (FIG. 1).

pLaci was generated by digesting pBluescript KS (Stratagene) with SacIand NaeI, blunt ending with T4 DNA polymerase, and religating. UL53,UL55, and kanamycin sequences were amplified by PCR using primerscontaining PvuII, HindIII, and KpnI/XhoI sites, respectively (Table 1).The UL53 fragment was digested with PvuII and cloned into the uniquePvuII site of pLaci, creating UL53-pLaci. The orientation of thefragment was confirmed by restriction enzyme digestion. Likewise, theUL55 fragment was digested with HindIII and cloned into the HindIII siteof UL53-pLaci, creating UL53-pLaci-UL55. The orientation was confirmedby restriction digestion. The kanamycin fragment was digested with KpnIand XhoI, and cloned into KpnI/XhoI-digested UL53-pLaci-UL54. Thisresults in an in-frame fusion of lacZ α-complementation and kanamycinsequences. This plasmid was called UL53-pLaci-kan-UL55. pHSV-GFP (GreenFluorescent Protein) was constructed by replacing lacZ sequences inpHSVLac (Geller, supra) with GFP sequences.

Polymnerase Chain Reaction (PCR)

PCR-amplified fragments were obtained using viral genomic DNA or plasmidDNA as template and the oligonucleotides listed in Table 1 as primers.Taq DNA polymerase (Promega) was used to amplify the region of interest,according to the recommendations of Sambrook et al. (Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and 30 cycles (95° C., 1 minute, 55° C., 1 minute,72° C., 1 minute) were carried out in a Perkin Elmer GeneAmp 2400.

Virus Construction

Plasmid BAC-TK was digested with HindIII and co-transfected into Verocells with infectious HSV-1 F strain DNA, as described by Chiou et al.(Virology 145(2):213-226, 1985). DNA from plaques that were resistant to100 μM acyclovir (ACV) was screened by PCR using primers that correspondto chloramphenicol sequences (Table 1). From each of two independenttransfections, one recombinant virus was plaque-purified two times andthe mutation confirmed by Southern blot hybridization. This virus wasdesignated HSV-BAC.

Isolation of HSV-BAC DNA and BAC Plasmids

One hundred mm dishes of confluent Vero cells were infected with HSV-BACat an m.o.i. of 3. Infections were allowed to proceed for two hours, thesupernatants were removed, and 1 ml of DNAzole (Gibco) was added. DNA,obtained from the cell lysate following the manufacturer's protocol, wasresuspended in 100 μl of TE. One μl of DNA was added to an equal volumeof water and electroporated into 25 μl of electro-competent E. coli,strain DH 10B (Hanahan et al., Methods Enzymol. 204:63-113, 1991) usinga cell-porator (Gibco) and conditions recommended by the manufacturer.The bacteria were allowed to recover by incubation at 37° C. in LBbroth, plated out onto LB agar plates containing chloramphenicol (12.5ng/ml), and incubated at 37° C. overnight. BAC plasmids were isolatedfrom E. coli using an alkaline-lysis procedure and screened by PCR. Foursets of primers were used to amplify sequences in US6, UL10, UL30, andUL40 (Table 1). Two μg of DNA from clones that were positive for allprimers sets was transfected into a 100 mm dish of Vero cells using 18μl of lipofectamine, and incubated at 34° C. Viral plaques were observedtwo days post-transfection.

BAC Mutagenesis

p25-containing bacteria were made competent by CaCl₂ treatment.pUL53-pLaci-kan-UL55 was digested with XbaI, M1uI, and ScaI, and theUL53-pLaci-kan-UL55 sequences were gel purified and transformed intocompetent p25 E. coli. The bacteria were allowed to recover for 2 hoursat 37° C. in the presence of 1 mM IPTG, then plated out onkanamycin/IPTG plates (5 mg/ml and 1 mM, respectively). Mutations wereconfirmed by PCR and Southern blot hybridization. Mutant viruses weretested for growth on 2—2 and Vero cells.

Amplicon Production

2—2 cells were transfected with 1, 2, or 4 μg of either p25, HSV cosmidset 6, 28, 14, 56, and 48 (Cunningham et al., supra), or infectious HSVF strain DNA and 1 g of pHSV-GFP, as described by Fraefel et al. (J.Virol. 70(10):7190-7197, 1996). Cells were harvested after four days,frozen and thawed three times, the cellular debris was removed bycentrifugation, and the supernatant was used to infect fresh monolayers.Titers were calculated by counting viral plaques and transduced greencells.

TABLE 1 Primers Chloramphenicol 5′-AGGCCGGATAGCTTGTGC-3′ (SEQ ID NO:1)5′-CGGAACAGAGAGCGTCACA-3′ (SEQ ID NO:2) Kanamycin5′-GGATGAGGATCGGTACCCATGATTGAAC-3′ (SEQ ID NO:3)5′-ATACTCATACTCGAGCTCTTCCTTTTTC-3′ (SEQ ID NO:4) US65′-CCGAATGCTCCTACAACAAG-3′ (SEQ ID NO:5) 5′-GTCTTCCGGGGCGAGTTCTG-3′ (SEQID NO:6) UL10 5′-GGTGTAGCCGTGCCCCTCAG-3′ (SEQ ID NO:7)5′-GCAGATACGTCCCGCTCAGG-3′ (SEQ ID NO:8) UL305′-ATCAACTTCGACTGGCCCTTC-3′ (SEQ ID NO:9) 5′-CCGTACATGTCGATGTTCACC-3′(SEQ ID NO:10) UL40 5′-ACCGCTTCCTCTTCGCTTTC-3′ (SEQ JD NO:11)5′-CCCGCAGAAGGTTGTTGGTG-3′ (SEQ ID NO:12) UL535′-CCTCTCGGGCACAGCTGTGCGATTGTG-3′ (SEQ ID NO:13)5′-AAGCGTGACCGCAGCTGGAACACGGCTG-3′ (SEQ ID NO:14) UL555′-CGTGTCTGCAAGCTTACGGCCAGTC-3′ (SEQ ID NO:15)5′-CCATGCCGCTGAAGCTTATGGAGCGCAGG-3′ (SEQ ID NO:16)

TABLE 1 Primers Chloramphenicol 5′-AGGCCGGATAGCTTGTGC-3′ (SEQ ID NO:1)5′-CGGAACAGAGAGCGTCACA-3′ (SEQ ID NO:2) Kanamycin5′-GGATGAGGATCGGTACCCATGATTGAAC-3′ (SEQ ID NO:3)5′-ATACTCATACTCGAGCTCTTCCTTTTTC-3′ (SEQ ID NO:4) US65′-CCGAATGCTCCTACAACAAG-3′ (SEQ ID NO:5) 5′-GTCTTCCGGGGCGAGTTCTG-3′ (SEQID NO:6) UL10 5′-GGTGTAGCCGTGCCCCTCAG-3′ (SEQ ID NO:7)5′-GCAGATACGTCCCGCTCAGG-3′ (SEQ ID NO:8) UL305′-ATCAACTTCGACTGGCCCTTC-3′ (SEQ ID NO:9) 5′-CCGTACATGTCGATGTTCACC-3′(SEQ ID NO:10) UL40 5′-ACCGCTTCCTCTTCGCTTTC-3′ (SEQ JD NO:11)5′-CCCGCAGAAGGTTGTTGGTG-3′ (SEQ ID NO:12) UL535′-CCTCTCGGGCACAGCTGTGCGATTGTG-3′ (SEQ ID NO:13)5′-AAGCGTGACCGCAGCTGGAACACGGCTG-3′ (SEQ ID NO:14) UL555′-CGTGTCTGCAAGCTTACGGCCAGTC-3′ (SEQ ID NO:15)5′-CCATGCCGCTGAAGCTTATGGAGCGCAGG-3′ (SEQ ID NO:16)

All references cited herein are incorporated by reference in theirentirety. Other embodiments are within the following claims.

16 18 base pairs nucleic acid single linear cDNA not provided 1AGGCCGGATA GCTTGTGC 18 19 base pairs nucleic acid single linear cDNA notprovided 2 CGGAACAGAG AGCGTCACA 19 28 base pairs nucleic acid singlelinear cDNA not provided 3 GGATGAGGAT CGGTACCCAT GATTGAAC 28 28 basepairs nucleic acid single linear cDNA not provided 4 ATACTCATACTCGAGCTCTT CCTTTTTC 28 20 base pairs nucleic acid single linear cDNA notprovided 5 CCGAATGCTC CTACAACAAG 20 20 base pairs nucleic acid singlelinear cDNA not provided 6 GTCTTCCGGG GCGAGTTCTG 20 20 base pairsnucleic acid single linear cDNA not provided 7 GGTGTAGCCG TGCCCCTCAG 2020 base pairs nucleic acid single linear cDNA not provided 8 GCAGATACGTCCCGCTCAGG 20 21 base pairs nucleic acid single linear cDNA not provided9 ATCAACTTCG ACTGGCCCTT C 21 21 base pairs nucleic acid single linearcDNA not provided 10 CCGTACATGT CGATGTTCAC C 21 20 base pairs nucleicacid single linear cDNA not provided 11 ACCGCTTCCT CTTCGCTTTC 20 20 basepairs nucleic acid single linear cDNA not provided 12 CCCGCAGAAGGTTGTTGGTG 20 27 base pairs nucleic acid single linear cDNA not provided13 CCTCTCGGGC ACAGCTGTGC GATTGTG 27 28 base pairs nucleic acid singlelinear cDNA not provided 14 AAGCGTGACC GCAGCTGGAA CACGGCTG 28 25 basepairs nucleic acid single linear cDNA not provided 15 CGTGTCTGCAAGCTTACGGC CAGTC 25 29 base pairs nucleic acid single linear cDNA notprovided 16 CCATGCCGCT GAAGCTTATG GAGCGCAGG 29

What is claimed is:
 1. A bacterial artificial chromosome constructcomprising a nucleic acid sequence that directs formation of arecombinant virus upon introduction into a cell.
 2. The artificialchromosome construct of claim 1, wherein said recombinant virus is alytic virus.
 3. The artificial chromosome construct of claim 1, whereinsaid recombinant virus is a non-lytic virus.
 4. The artificialchromosome construct of claim 1, wherein said artificial chromosome orsaid nucleic acid sequence further comprises a heterologous nucleic acidsequence.
 5. The artificial chromosome construct of claim 1, whereinsaid recombinant virus is a herpes virus.
 6. The artificial chromosomeconstruct of claim 4, wherein said heterologous nucleic acid sequenceencodes a therapeutic gene product.
 7. The artificial chromosomeconstruct of claim 6, wherein said therapeutic gene product is selectedfrom the group consisting of growth factors, hormones, enzymes, vaccineantigens, cytotoxins, immunomodulatory proteins, antisense RNAmolecules, and ribozymes.
 8. The artificial chromosome construct ofclaim 5, wherein said herpes virus is a herpes simplex virus.
 9. A cellcomprising a bacterial artificial chromosome construct stably integratedinto its genome.
 10. The cell of claim 9, wherein said artificialchromosome construct comprises a nucleic acid sequence that encodes anHSV genome in which an immediate early gene comprises a mutation.